Molecularly imprinted polymers, or MIPs, have become an area of tremendous scientific interest in the field of separations. Since the first publication of imprinting in an organic polymer three decades ago [G. Wulff, Agnew. Chem. Int. Ed. Engl., 11: 341 (1972)], MIPs have become recognized as suitable for a vast number of industrial applications. Molecular imprinting creates selective adsorptive or catalytic sites within an organic or inorganic polymer. Typically, this procedure involves polymerizing functional and crosslinking monomers in the presence of a template molecule that interacts with the functional monomer(s) via noncovalent or reversible covalent bonds. The template molecule forms imprints in the polymer. Extracting the template from the polymer leaves behind voids in the MIP where selective rebinding of the template or a template analog can occur. The imprinted polymer is then used as a specific separation tool with high selectivity or recovery for analytes (having identical or similar structure to the template molecule) from a sample. Given the tremendous diversity of suitable template molecules, a correspondingly vast array of molecularly imprinted polymers can be created for any given separation or catalytic application.
For example, the synthesis and use of molecularly imprinted polymers for applications such as solid phase adsorbents, chiral stationary phases, sensors, and even as weak enzyme mimics has been reviewed by a variety of investigators (see, for instance, O. Bruggemann et al., J. Chromatography A, 889: 15 (2000)).
The benefits of MIP-based separations include the following: 1) MIPs can be created to have high affinity and selectivity for virtually any particular molecule desired; 2) MIPs have a unique stability that is superior to that of natural biomolecules (e.g., antibodies or proteins which can also selectively rebind); 3) MIPs, or “antibody mimics”, can operate in extreme conditions not appropriate for use of antibodies (e.g., at elevated temperatures, in organic solvents, and at extreme pH); 4) MIPs have high binding capabilites (e.g., up to several μM concentrations can normally be extracted using 5-20 mg of sorbent); and 5) MIPs are relatively simple and inexpensive to prepare.
However, adsorbent or catalytic MIPs suffer from two major deficiencies that render them unsuitable for widespread industrial use. First, the mass transfer performance of MIPs is low, a cause of poor adsorbate access to the imprinted sites. Secondly, the low adsorption capacity for adsorbent MIPs and the low catalytic rates for catalytic MIPs, respectively, limit their utility. Poor monomer-template interactions are recognized as the underlying cause of these deficiencies that prevent successful molecular imprinting. Frequently, poor monomer-template interactions result from interference created by the suspension medium [see, for instance, P. Cormack and K. Mosbach, React. Funct. Polym. 41(1-3), 115-124 (1999); K. Mosbach and K. Haupt, J. Mol. Recognit. 11(1-6), 62-68 (1998); and G. Wulff, Chemtech, 28:19 (1998)].
Previously, MIPs have been prepared by three primary techniques: bulk polymerization, suspension polymerization, and surface polymerization. These three techniques are described below, in terms of their respective method of synthesis and primary disadvantages.
Imprinting via bulk polymerization typically occurs by mixing together template, monomers, and initiators directly in a solvent, and then permitting polymerization to occur. This process creates chunks of polymer which must then be crushed, ground, and sieved to obtain the desired particle size [L. Andersson et al., Chromatographia, 46:57-62 (1997); B. A. Rashid et al., Analytical Communications, 34:303-5 (1997)]. In this method monomer-template interactions are expected to occur successfully in the presence of the solvent, and no mechanism exists to control particle morphology. Instead, grinding generates irregularly sized particles that pack poorly into a column and can potentially fracture the poorly formed active sites, further reducing or eliminating their usability. Grinding also generates a large amount of undesirable fines, which can exceed 50% of the original polymer mass. For these reasons, bulk polymerization is inherently inefficient, particularly at larger scale [O. Bruggemann et al., J. Chromatography A, 889:15-24 (2000); U.S. Pat. No. 5,959,050].
Imprinting via suspension polymerization uses a two-phase agitated system. Typically, monomers, template, and initiator form a first phase, while the suspending medium (water or another highly polar liquid) acts as a second phase. Polymerizing conditions are applied (e.g., heat or ultraviolet radiation) and the monomers are polymerized while they are dispersed throughout the second phase by agitation. However, water can interfere with monomer-template interactions through strong hydrogen bonding with the template. Because a large excess of water is typically present, it can actually saturate the monomer phase, disrupting the desired monomer-template interactions. The large excess of water or other suspending media can also solubilize the template, removing it from the monomer phase. This condition lowers the theoretical efficiency of imprinting (a ratio of the mass of template adsorbed relative to the mass of template used during polymerization). As a result, MIPs made with this method typically do not perform satisfactorily [B. Sellergren, J. Chromatography A, 673: 133 (1994)].
Imprinting via supension polymerization is exemplified in WO 00/41723, WO 00/41723 discloses a surfactant-free means of making molecularly imprinted particles ranging in size from 0.01-10 microns. However, the monomer-template interactions take place within a large excess of suspending media. In addition, the polymer throughput of this system is quite low due to the limit of 0.01-20% volume of polymerizable compounds relative to suspending media. U.S. Pat. No. 5,872,198 and U.S. Pat. No. 5,959,050 also disclose imprinting via supension polymerization; in these patents, however, perfluorocarbons are used as the suspending media in order to avoid the use of water. The primary disadvantage of perfluorocarbons is that they generally require a fluorinated surfactant, thus requiring capital-intensive solvent recovery systems. Further, the high density of perfluorocarbons (e.g., perfluoro[cyclohexane] density=1.78 grams/milliliter) requires significant power input to prevent bulk phase separation during suspension polymerization. Finally, such perfluorocarbons can be environmentally unfriendly and present handling and disposal problems.
The third method of molecular imprinting is referred to as imprinting via surface polymerization [J. Haginaka et al., J. Chromatography A, 849: 331-9 (1999)]. In this process, a small coating of monomer and template is permitted to polymerize onto a pre-existing polymer surface. Such a method is inherently undesirable for many industrial applications because only the polymer surface is imprinted (the remainder is wasted or leads to nonspecific binding). As a result, the method requires large quantities of polymer since much of the polymer does not contain the adsorption sites of interest. In addition, when water is used as the continuous or second phase, template leaching and poor monomer-template interactions may interfere with the imprinting process.
Currently, the literature reveals no mention of a suspension polymerization method that allows these critical monomer-template interactions to occur without disruption. Neither is a general process described where a monomer-template mixture is allowed to associate to establish imprinted sites in the absence of suspending media, followed by a rapid polymerization to achieve good particle morphology and size control. Similarly, there is no art disclosing the concept of the imprinting and the extracting of a template in the same vessel—a useful improvement to process operation at the industrial level.
In the broad field of polymer synthesis (not including MIPs), methods are known in which a monomer mixture is premixed and allowed to form individual particles, then introduced into an aqueous phase where polymerization fully occurs. U.S. Pat. No. 3,922,255 discloses jetting a monomer mixture into a column-shaped vessel containing an aqueous phase under convective flow, followed by the transfer of the monomer/aqueous reaction mixture to a separate column maintained at elevated temperature via an aqueous feed, so as to allow polymerization of the monomer droplets. However, this method is not for formation of MIPs and considers neither template molecules nor the need for template-monomer interactions for molecular imprinting.
Thus, a need exists for a facile and reproducible polymerization method that permits formation of MIPs possessing improved binding affinity (in the form of strong template-monomer interactions unimpeded by solvent), uniform particle size distribution, and good mass transfer performance. The method needs to be simple and cost effective. Additionally, the method must eliminate: 1) the need for grinding of the polymer; 2) requirements for use of exotic fluids; and 3) premature loss of template to the suspending liquid. Such a method would specifically solve the problem of how to achieve efficient separation of isoflavones from soy whey.